Primescript rt master mix

Total RNA from cells or tissues was extracted using TRIzol^® reagent (Invitrogen, USA). Isolated RNA was reverse-transcribed and duplicated using PrimeScript™ RT Master Mix (Vazyme, Nanjing, China) and SYBR qPCR master mix (Vazyme, Nanjing, China) in iScript cDNA Synthesis Kit (Takara BIO, Otsu, Japan) and the Light-Cycler 480 Real-Time PCR System (Roche, San Francisco, CA, USA). Primers sequences are listed in Supplementary Table S1. The results were normalized to GAPDH and expressed as percentage of controls.

Total RNA samples were extracted by TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and reversed transcription was performed according to the protocol using PrimeScript RT Master Mix (Vazyme, Nanjing, China). Consequently, the qPCR was conducted with the SYBR Green kit (Applied Biosystems) and the Step One Plus PCR system (Applied Biosystems).
The primer sequences are shown below:

IL‐1β:

Forward, CCATCCTCTGTGACTCATGGG;

Reverse, TCAGCTCATATGGGTCCGAC;

IL‐6:

Forward, GACAAAGCCAGAGTCCTTCAGAGAG;

Reverse, CTAGGTTTGCCGAGTAGATCTC;

TNF‐α:

Forward, CCACCACGCTCTTCTGTCTA;

Reverse, GATCTGAGTGTGAGGGTCTGG;

iNOS:

Forward, CACCACCCTCCTCGTTC;

Reverse, TGCCTATCCGTCTCGTC;

COX‐2:

Forward, TGCTGGTGGAAAAACCTCGT;

Reverse, AAAACCCACTTCGCCTCCAA;

CD86:

Forward, CCAGAACTTACGGAAGCACC;

Reverse, CCAGAACACACACAACGGTC;

Arg‐1:

Forward, AGCACTGAGGAAAGCTGGTC;

Reverse, TACGTCTCGCAAGCCAATGT;

GAPDH:

Forward, AGGTCGGTGTGAACGGATTTG;

Reverse, TGTAGACCATGTAGTTGAGGTCA.

Total RNA was isolated from the indicated cells or tissues using Trizol reagent (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. cDNA was synthesized using PrimeScript RT Master Mix (#R323-01, Vazyme, China), and qRT-PCR was performed using a SYBR Green Master Mix (#Q711-02, Vazyme, China). GAPDH expression was used as an endogenous control. The primer used is shown in Supplementary Table S2.

After treatment with the indicated inhibitors, cells were washed with PBS and then lysed with TRIzol reagent (Invitrogen, #15596018) for RNA extraction. After the removal of genomic DNA, mRNAs were used as templates to synthesize cDNAs using PrimeScript RT Master Mix (Vazyme, #R323‐01). The cDNAs were then used as templates for RT‐qPCR analysis using SYBR Green qPCR Master Mix (Vazyme, #Q711‐03) to detect the expression levels of target genes. GAPDH was used as an internal control. Primer sequences were obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/) and are shown in Table S5.

Prior to commencing the study, total RNA was extracted from PANC-1 and MIA-PaCa-2 cells using the TRIzol® reagent (Vazyme Biotech Co., Ltd). The concentration and purity of RNA were then measured using NanoDrop 2000 equipment (Invitrogen, USA). Once the total RNA was extracted, we conducted RT-PCR using the PrimeScript RT Master Mix (Vazyme Biotech Co., Ltd) with corresponding primers to get the complementary DNA (cDNA) of target circRNAs and mRNAs. While the cDNA of target miRNAs was generated using the PrimeScript RT Reagent Kit (Vazyme Biotech Co., Ltd) with specific stem-loop primers. Then, the indicating cDNA was checked by qPCR using the Real-Time PCR Master Mix (SYBR Green; Vazyme Biotech Co., Ltd) in the CFX Connect Fluorescent Quantitative PCR Instrument (Bio-Rad, USA). Different primers were used in our study; U6 was used as the internal reference standard for miR-139-5p, and GAPDH was used as the internal reference standard for circRNF13 and IGF1R. All the primers used are listed below:

 
β-actin, forward: GTCTGCCTTGGTAGTGGATAATG and reverse: TCGAGGACGCCCTATCATGG

  IGF1R, forward: TCGACATCCGCAACGACTATC and reverse: CCAGGGCGTAGTTGTAGAAGAG

  CircRNF13, forward: CCTTATCATAGTGGGCATCTGTC and reverse: AGCATCTCGTTGTAAAATCACCTT

  miR-139-5p, forward: ACACTCCAGCTGGGGACCTCTGTGCACGTG and reverse: TGGTGTCGTGGAGTCG

  U6, forward: CTCGCTTCGGCAGCACA and reverse: AACGCTTCACGAATTTGCGT

Total RNA was isolated from cells using 0.5 mL Trizol, and reverse transcription quantitative PCR was performed using the Prime-Script RT Master Mix (Vazyme, China). qRT-PCR was performed using SYBR Green Realtime PCR Master Mix (Vazyme, China). Samples from each experiment were independently repeated three times. The sequences were as follows: CDCA8: Forward, TTGACTACTTCGCCCTTG, Reverse, CTTCTTCTTCCTCTTCCACTA; TPM3: Forward, 5′-GCACATTGCA GAAGAGGCAG-3′; Reverse, 5′-TCTGTGCGTTCCAAGTCTCC-3′; USP13: Forward, 5′-GCCAAGCACTTAGCGCATTT-3′; Reverse, 5′-CACTTCCCACTCA CTGACCC-3′; NECAP2: Forward, 5′-AACAAGCCCAGAACCCAGAC-3′, Reverse, 5′-CCAGCTGCTCCTTCCTTCTT-3’; SPRYD4: Forward, 5′-CAAGCTGGGGAAC AGCCATA-3′, Reverse, 5′-GAATTTCTGCCCCTTCACGC-3′.
The primary antibodies used in this study were as follows: anti-CDCA8 (Santa Cruz, sc-376635), anti-NF-YA (Santa Cruz, sc-17753), anti-MEK1/2 (CST, 4694), anti-Phospho-MEK1/2 (CST, 9154), anti-p44/42 (CST, 4695), anti-Phospho-p44/42 (CST, 4377), anti-p38 (CST, 8690), anti-rabbit IgG (CST, 4413), and anti-mouse IgG (CST, 7076).

Anti-BMAL1 (ab3350), anti-H3K27ac (ab4729), anti-H3K4me3 (ab8580), anti-H3K9me3 (ab8898) and anti-GAPDH (ab8245) antibodies were purchased from Abcam (Cambridge, UK). Anti-REV-ERBα (14506-1-AP), anti-DBP (12662-1-AP), anti-SLC1A5 (20350-1-AP), and anti-Histone H3 (17168-1-AP) antibodies were purchased from Proteintech (Wuhan, China). Anti-PPAR-γ (2443), anti-H3K9ac (9649), anti-H3K9me2 (4658), anti-H3K27me3 (9733) and anti-rabbit IgG (2729) were purchased from Cell Signaling Technology (Danvers, MA). Glutamine, methionine, C646 and MM-102 were purchased from Tsbiochem (Shanghai, China). [¹³C₅]-glutamine and [²H₃]-methionine were purchased from Zzbio (Shanghai, China). Dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO). RNAiso Plus reagent, PrimeScript RT Master Mix and ChamQ Universal SYBR qPCR Master Mix were purchased from Vazyme (Nanjing, China). Beetle luciferin was purchased from Promega (Madison, WI). siRNAs for Slc1a5 and Ppar-γ, and negative control were purchase from IGE Biotechnology (Guangzhou, China). pcDNA 3.1, pcDNA 3.1-Ppar-γ, pcDNA 3.1-Slc1a5, pRL-TK, pGL4.0 and Slc1a5 luciferase reporters (-1400/+100 bp, -1000/+100 bp and a PPRE-mutated version) were obtained from Kaile Technologies (Guangzhou, China).